Hydroxyethylstarch

ABSTRACT

Described are a hydroxyethylstarch, a process for the preparation thereof, a pharmaceutical formulation containing such a hydroxyethylstarch, and the use of the pharmaceutical formulation for the preparation of a volume replacement, a plasma replacement or a plasma volume expander, as well as the use of the pharmaceutical formulation for maintaining normovolemia and/or for improving the macro- and microcirculation and/or for improving the nutritive oxygen supply and/or for stabilizing hemodynamics and/or for improving the volume efficiency and/or for reducing the plasma viscosity and/or for increasing anemia tolerance and/or for hemodilution, especially for therapeutic hemodilution in disturbed blood supply and arterial, especially peripheral arterial, occlusive diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a US national phase application of PCT/EP2005/050877, filed Mar.1, 2005, which claims priority to a German Registration No. 04100813.7,filed Mar. 1, 2004, the contents of each of which are expresslyincorporated herein by reference.

The invention relates to a hydroxyethylstarch, and to a process for thepreparation of such a hydroxyethylstarch. Further, the invention relatesto a pharmaceutical formulation containing a hydroxyethylstarch, and tothe use of such a pharmaceutical formulation for the preparation of avolume replacement, a plasma replacement or a plasma expander, as wellas to the use of the pharmaceutical formulation for maintainingnormovolemia and/or for improving the macro- and microcirculation and/orfor improving the nutritive oxygen supply and/or for stabilizinghemodynamics and/or for improving the volume efficiency and/or forreducing the plasma viscosity and/or for increasing anemia toleranceand/or for hemodilution, especially for therapeutic hemodilution indisturbed blood supply and arterial, especially peripheral arterial,occlusive diseases.

BACKGROUND

The use of intravascular fluid is among the most important measures inthe prophylaxis and therapy of hypovolemia, irrespective of whether thehypovolemia results from the immediate loss of blood or body fluids (inacute bleedings, traumas, surgery, burns), from disturbed distributionbetween macro- and microcirculation (such as in sepsis), or fromvasodilation (e.g., during the initiation of anesthesia). Infusionssuitable for such indications are supposed to restore normovolemia andmaintain the perfusion of vital organs and the peripheral blood flow. Atthe same time, the solutions must not stress the circulationexcessively, and they must be possibly free from side effects. In thisrespect, all the currently available volume replacements have benefitsand drawbacks. Although so-called crystalloid solutions (electrolytesolutions) are essentially free from immediate side effects, they ensureonly a short-term or inadequate stabilization of the intravascularvolume and the hemodynamics. In case of pronounced or persistinghypovolemia, they must be infused in excessive amounts because they donot exclusively remain in the intravascular compartment but quicklydissipate in the extravascular space. However, a fast flow-out into theextravascular space not only limits the circulation-filling effect ofcrystalloid solutions but also involves the risk of peripheral andpulmonary edemas. Apart from the vital threat which a lung edema canrepresent, it additionally leads to a deterioration of the nutritiveoxygen supply, which is also affected by peripheral edemas.

In contrast, colloidal volume replacements, whether the colloidscontained therein are of natural or synthetic origin, have a much morereliable effect. This is due to the fact that, because of theircolloid-osmotic effect, they retain the supplied liquid longer in thecirculation as compared to crystalloids and thus protect them fromflowing out into the interstice. On the other hand, colloidal volumereplacements cause a higher extent of undesirable responses as comparedto crystalloid solutions. Thus, the natural colloid albumin, like allblood or plasma derivatives, involves the risk of infection with viraldiseases; in addition, it may result in interactions with other drugs,e.g., ACE inhibitors; finally, the availability of albumin is limited,and its use as a volume replacement is disproportionately expensive.Further doubts as to the use of albumin as a volume replacement are dueto the inhibition of the endogenous synthesis of albumin if it is addedexogenously and due to its ready extravascularization. This means thepassage from the circulation into the extravascular space, whereundesirable and persistent liquid accumulations can occur because of thecolloid-osmotic effect of albumin.

In the synthetic colloids, severe anaphylactoid responses and a massiveinhibition of blood coagulation have caused dextran preparations todisappear almost completely from therapy. Although hydroxyethylstarch(HES) solutions also have the potential for triggering anaphylactoidresponses and affecting blood coagulation, this is to a lesser extent ascompared with dextran. Severe anaphylactoid responses (responses ofseverity III and IV) are observed extremely rarely with HES solutions,in contrast to dextran, and the influence on blood coagulation, inherentto the high-molecular weight HES solutions, could be significantlyreduced in recent years by the further development of HES solutions. Ascompared with gelatin solutions, which also find use as plasmareplacements and leave blood coagulation essentially unaffected, HESsolutions, at least their high- and medium-molecular weight embodiments,have the benefit of a longer plasma residence time and effectiveness.

EP-A-0 402 724 discloses the preparation and use of a hydroxyethylstarchhaving an average molecular weight, Mw, of from 60,000 to 600,000, amolar substitution, MS, of from 0.15 to 0.5, and a degree ofsubstitution, DS, of from 0.15 to 0.5. The disclosure deals with therapid (6 to 12 hours) and complete degradability of thehydroxyethylstarches to be employed as plasma expanders. Within thepreferred range of average molecular weights of from 100,000 to 300,000,a hydroxyethylstarch having an average molecular weight of 234,000 wasexplicitly examined.

U.S. Pat. No. 5,502,043 discloses the use of hydroxyethylstarches havingan average molecular weight, Mw, of from 110,000 to 150,000, a molarsubstitution, MS, of from 0.38 to 0.5, and a degree of substitution, DS,of from 0.32 to 0.45 for improving microcirculation in peripheralarterial occlusive disease. In addition, the document teaches the use oflow-molecular weight (Mw 110,000 to 150,000) hydroxyethylstarches which,due to their low molecular weight, keep the plasma viscosity low andthus ensure an improvement of microcirculation in the blood flow.However, this document advises against the use of higher-molecularweight hydroxyethylstarches, such as a hydroxyethylstarch with an Mw of500,000, because they increase plasma viscosity and thus deterioratemicrocirculation despite their low molar substitution (MS=0.28).

Worldwide, different HES preparations are currently used as colloidalvolume replacements, which are mainly distinguished by their molecularweights and additionally by their extent of etherification withhydroxyethyl groups, and by other parameters. The best knownrepresentatives of this class of substances are the so-called Hetastarch(HES 450/0.7) and Pentastarch (HES 200/0.5). The latter is the currentlymost widespread “standard HES”. Besides, HES 200/0.62 and HES 70/0.5play a minor role. The declared information relating to the molecularweight as well as that relating to the other parameters are averagedquantities, where the molecular weight declaration is based on theweight average (Mw) expressed in Daltons (e.g., for HES 200,000) ormostly abbreviated in Kilodaltons (e.g., for HES 200). The extent ofetherification with hydroxyethyl groups is characterized by the molarsubstitution MS (e.g. as 0.5 such as in HES 200/0.5; MS=average molarratio of hydroxyethyl groups to anhydroglucose units) or by the degreeof substitution (DS=ratio of mono- or polyhydroxyethylated glucoses tothe total anhydroglucose units). According to their molecular weights,the HES solutions in clinical use are classified into high-molecularweight (450 kD), medium-molecular weight (200-250 kD) and low-molecularweight (70-130 kD) preparations.

As to the coagulation effects of HES solutions, a distinction is to bemade between non-specific and specific influences. A non-specificinfluence on blood coagulation results from dilution of the blood(hemodilution), which occurs during the infusion of HES solutions andother volume replacements into the circulation. Affected by thishemodilution are also coagulation factors, whose concentrations aredecreased depending on the extent and duration of the dilution of theblood and the plasma proteins due to the infusion. Correspondingly largeor persisting effects may result in a hypocoagulability which isdetectable by laboratory diagnostics and, in extreme cases, clinicallyrelevant.

In addition, hydroxyethylstarch may cause a specific influence on bloodcoagulation, for which several factors are held responsible. Thus, undercertain conditions or with certain HES preparations, a decrease of thecoagulation proteins factor VIII (F VIII) and von Willebrand factor(vWF) is found which is larger than the general decrease of the plasmaproteins due to hemodilution. Whether this larger than expected decreaseis caused by a reduced formation or release of F VIII/vWF, such as bycoating effects on the vascular endothelium caused by HES, or by othermechanisms is not quite clear.

However, HES influences not only the concentration of the coagulationfactors mentioned but evidently also the function of platelets. This iscompletely or in part due to the binding of HES to the surface of theplatelets, which inhibits the access of ligands to the fibrinogenreceptor of the platelets.

These specific effects of HES on blood coagulation are particularlypronounced when high-molecular weight HES (e.g., HES 450/0.7) areemployed while they do not play such a great role for medium-molecularweight (e.g., HES 250/0.5) or low-molecular weight HES (e.g., HES130/0.4 or HES 70/0.5) (J. Treib et al., Intensive Care Med. (1999), pp.258 to 268; O. Langeron et al., Anesth. Analg. (2001), pp. 855 to 862;R. G. Strauss et al., Transfusion (1988), pp. 257-260; M. Jamnicki etal., Anesthesiology (2000), pp. 1231 to 1237).

If the risk profile of high-molecular weight HES is compared with thatof the medium- and low-molecular weight preparations, a clear reductionof the risks can be established in the latter, i.e., not only withrespect to the interaction with blood coagulation but also with respectto particular pharmacokinetic properties. Thus, the high-molecularweight HES solutions show a high accumulation in the circulation whilethis drawback is reduced in medium-molecular weight HES and virtuallyabsent in low-molecular weight preparations. The fact that no moreaccumulation occurs with low-molecular weight HES solutions, such as HES130/0.4, is a relevant therapeutic progress because the plasma levels ofHES cannot be determined in clinical routine, and therefore, evenextreme concentrations, which can be obtained within a few days with thehigh-molecular weight solutions, remain undiscovered. In this case, theamount of “residual HES” accumulated in the circulation is unknown tothe user but it nevertheless influences the kinetics and behavior of theHES which was additively infused, not knowing the amounts still presentin the circulation. Therefore, the effect of high-molecular weight HESaccording to the prior art is not calculable; it remains longer in thecirculation than would be required or desired for therapeutic reasons inmost cases, and its metabolic fate is unclear.

In contrast, low-molecular weight HES will disappear completely from thecirculation within about 20 to 24 hours after the infusion. This avoidsbacklog effects, and no accumulation occurs, especially for repeatedinfusions. The pharmacokinetic behavior of low-molecular weight starch,in contrast to high-molecular weight starch, is calculable and thereforecan be easily controlled. Too high a load on the circulation or theclearance mechanisms does not occur.

However, this behavior of low-molecular weight HES as compared tohigh-molecular weight preparations, which is advantageous as such, ispurchased at the expense of a significantly shorter plasma half life.The plasma half life of low-molecular weight HES is only about half thatof HES 200 or less (J. Waitzinger et al., Clin. Drug Invest. (1998), pp.151 to 160) and is in the range of the half life of gelatinpreparations, which are to be rated as decidedly short-term effective.Although a short half life of a volume replacement need not becategorically disadvantageous, because it can be compensated for by amore frequent or more highly dosed administration of the volumereplacement in question, in severe or persisting hypovolemia, a volumereplacement with a short half life and short effective period involvesthe risk of insufficient circulation filling (much like with crystalloidsolutions) or, when the dosage is correspondingly increased forcompensating for this drawback, the risk of interstitial liquidoverload.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the course of the concentration of low-molecular weight HESand high-molecular weight HES in the plasma after infusion over time.

DETAILED DESCRIPTION

Before this background, there is a need for a volume replacement whichon the one hand is characterized by a low tendency to accumulation and alow influence on blood coagulation (such as low-molecular weight HES)but on the other hand has a longer half life as compared to thelow-molecular weight HES solutions, whose properties are close to thoseof crystalloid solutions.

Searching for a hydroxyethylstarch having such properties, it has nowbeen found that there very well is a hydroxyethylstarch for HESsolutions with longer plasma half lives as compared to knownlow-molecular weight HES solutions, and that these can also be preparedand, surprisingly, without these high-molecular weight solutionsaccording to the invention having the drawbacks of previoushigh-molecular weight solutions, such as their property to becomeaccumulated in the circulation or their pronounced inhibition of bloodcoagulation.

Therefore, in one embodiment, the invention relates to ahydroxyethylstarch having an average molecular weight, Mw, of greaterthan or equal to 500,000, the molar substitution MS being from 0.25 to0.5, preferably from 0.35 to 0.50 (0.35≦MS≦0.50), and the C₂/C₆ ratiobeing from 2 to below 8.

The hydroxyethylstarches according to the invention are influenced bythe molar substitution MS. The molar substitution MS is defined as theaverage number of hydroxyethyl groups per anhydroglucose unit(Sommermeyer et al., Krankenhauspharmazie (1987), pp. 271 to 278). Themolar substitution can be determined according to Ying-Che Lee et al.,Anal. Chem. (1983) 55, 334, and K. L. Hodges et al., Anal. Chem. (1979)51, 2171. In this method, a known amount of HES is subjected to ethercleavage by adding adipic acid and hydroiodic acid (HI) in xylene.Subsequently, the ethyl iodide released is quantified by gaschromatography using an internal standard (toluene) and externalstandards (ethyl iodide calibrating solutions). The molar substitutionMS influences the effect of the hydroxyethylstarches according to theinvention. If the MS is selected too high, this may cause anaccumulation effect in the circulation when the hydroxyethylstarches areemployed as a volume replacement. On the other hand, if the MS isselected too low, this may result in too rapid a degradation of thehydroxyethylstarch in the circulation and thus reduce the desiredduration of the plasma half life. A molar substitution MS of from 0.35to 0.5 (0.35≦MS≦0.50), preferably from 0.39 to smaller than or equal to0.45 (0.39≦MS≦0.45) and especially an MS of from greater than 0.4 to0.44 (0.4<MS≦0.44), has proven advantageous.

The hydroxyethylstarches according to the invention belong to thehigher-molecular weight hydroxyethylstarches and preferably have anaverage molecular weight (Mw) of above 600,000 to 1,500,000, morepreferably from 620,000 to 1,200,000, especially from 700,000 to1,000,000. Due to the preparation conditions, the hydroxyethylstarchesare not in the form of a substance with a defined uniform molecularweight but in the form of a mixture of molecules of different sizeswhich are also differently substituted by hydroxyethyl groups.Therefore, the characterization of such mixtures requires recourse tostatistically averaged quantities. Therefore, the weight-averagemolecular weight (Mw) serves for characterizing the average molecularweight, the general definition of this mean value being stated inSommermeyer et al., Krankenhauspharmazie (1987), pp. 271 to 278.

The molecular weight determination can be effected by means of GPC-MALLSusing the GPC columns TSKgel G 6000 PW, G 5000 PW, G 3000 PW and G 2000PW (7.5 mm×30 cm), the MALLS detector (DAWN-EOS; Wyatt Deutschland GmbH,Woldert) and the RI detector (Optilab DSP; Wyatt Deutschland GmbH,Woldert) at a flow rate of 1.0 ml/minute in a 50 mM phosphate buffer, pH7.0. The evaluation may be performed by means of ASTRA software (WyattDeutschland GmbH, Woldert).

Preferred are those hydroxyethylstarches which are obtainable fromnative or partially hydrolyzed cereal or potato starches. Due to theirhigh content of amylopectin, the use of starches from waxy varieties ofthe corresponding crops, if they exist (e.g., waxy maize, waxy rice), isparticularly advantageous.

The hydroxyethylstarch according to the invention is further describedby the ratio of substitution at C₂ to substitution at C₆ of theanhydroglucose units. This ratio, which is also abbreviated as C₂/C₆ratio within the scope of this invention, means the ratio of the numberof anhydroglucose units substituted in 2 position to the number ofanhydroglucose units substituted in 6 position of thehydroxyethylstarch. The C₂/C₆ ratio of an HES can be varied widely bythe amount of aqueous sodium hydroxide used in the hydroxyethylation, asshown in Tables 1 and 2. The higher the amount of NaOH employed, themore highly the hydroxy groups in 6 position in the anhydroglucose ofthe starch are activated for hydroxyethylation. Therefore, the C₂/C₆ratio decreases during the hydroxyethylation with increasing NaOHconcentration. The determination is effected as stated by Sommermeyer etal., Krankenhauspharmazie (1987), pp. 271 to 278. With increasingpreference in the order given, the C₂/C₆ ratios are preferably from 3 tobelow 8, from 2 to 7, from 3 to 7, from 2.5 to smaller than or equal to7, from 2.5 to 6, or from 4 to 6. In the higher-molecular weight HESaccording to the invention, the C₂/C₆ ratio is another contribution toachieving the objects of the invention.

Due to their excellent tolerability and ready degradability in the humanor animal organism, the hydroxyethylstarches according to the inventionare suitable for being employed in a wide variety of pharmaceuticalformulations.

In a particular embodiment, the HES according to the invention have anaverage molecular weight of from 700,000 to 1,000,000, a molarsubstitution of from above 0.4 to 0.44 (0.4<MS≦0.44), and a C₂/C₆ ratioof from 2 to 7, preferably from 3 to 7, and especially from 2.5 to 6.

The present invention further relates to a process for the preparationof hydroxyethylstarch, preferably a hydroxyethylstarch according to theinvention. Preferably, the process comprises the following steps:

-   -   (i) reacting water-suspended starch, preferably corn starch,        more preferably partially hydrolyzed, so-called thin boiling,        waxy maize starch, with ethylene oxide; and    -   (ii) partially hydrolyzing the starch derivative obtained with        acid, preferably hydrochloric acid, until the desired range of        average molecular weight of the hydroxyethylstarch has been        reached.

In principle, all known starches are suitable for the preparation of thehydroxyethylstarches according to the invention, mainly native orpartially hydrolyzed starches, preferably cereal or potato starches,especially those having a high content of amylopectin. In a particularembodiment of the process according to the invention, starches from waxyvarieties, especially waxy maize and/or waxy rice, are employed. In aparticular embodiment, the preparation of HES is effected by reactingwater-suspended cereal and/or potato starch, preferably thin boilingwaxy maize starch, with ethylene oxide. Advantageously, the reaction iscatalyzed by adding alkalizing agents, preferably alkali metalhydroxides, for example, sodium hydroxide or potassium hydroxide.Therefore, in a preferred embodiment of the process according to theinvention, an alkalizing agent, preferably sodium hydroxide, isadditionally added to the water-suspended starch. The alkalizing agentis added to the suspended starch preferably in such an amount that themolar ratio of alkalizing agent to starch is greater than 0.2,preferably from 0.25 to 1, especially from 0.3 to 0.8. Through the ratioof ethylene oxide to starch during the hydroxyethylation step, the molarsubstitution, i.e., the molar ratio of hydroxyethyl groups toanhydroglucose units, can be arbitrarily controlled over the desired MSrange. Preferably, the reaction between ethylene oxide and suspendedstarch is effected in a temperature range of from 30 to 70° C.,preferably from 35 to 45° C. Usually, any residues of ethylene oxide areremoved after the reaction. In a second step following the reaction, anacidic partial hydrolysis of the derivatized starch is effected.“Partial hydrolysis” means the hydrolysis of the alpha-glycosidicallyinterconnected glucose units of the starch. In principle, all acidsfamiliar to the skilled person can be employed for the acidichydrolysis, but preferred are mineral acids, especially hydrochloricacid. The hydrolysis may also be effected enzymatically usingcommercially available amylases.

In another preferred embodiment, the process according to the inventionadditionally comprises the steps of (iii) sterile filtration andoptionally (iv) ultrafiltration. If the above described filtrations areperformed in the process according to the invention, the acidic partialhydrolysis of the raw HES is effected to an average molecular weightwhich is slightly below the desired target molecular weight. Byultrafiltration, low-molecular weight reaction by-products, mainlyethylene glycol, can be removed, the average molecular weight slightlyincreasing due to the elimination of part of the low-molecular weightHES fraction.

Preferably, the solutions derived from the preparation process aresubsequently diluted to the desired HES concentration, adjusted to thedesired osmotic pressure by adding salt, subjected to sterile filtrationand filled into suitable containers. Optionally, sterilization,preferably by live steam, can be effected.

Therefore, the present invention further relates to a pharmaceuticalformulation containing one or more hydroxyethylstarches according to theinvention. In principle, the pharmaceutical formulation according to theinvention can be provided in any possible galenic dosage form. In apreferred embodiment of the present invention, the pharmaceuticalformulations according to the invention can be injected or infusedintravenously. Therefore, the pharmaceutical formulations are preferablyin the form of an aqueous solution or colloidal aqueous solution.Preferably, the formulations contain the hydroxyethylstarches accordingto the invention in a concentration of up to 20%, more preferably from0.5 to 15%, more preferably from 2 to 12%, especially from 4 to 10%, forexample, 6%.

Unless stated otherwise, the amounts are expressed in %, which is to beunderstood as meaning g/100 ml of solution within the scope of thepresent invention.

In a further embodiment, the pharmaceutical formulations according tothe invention additionally contain sodium chloride, preferably from 0.6to 2%, more preferably 0.9%. A 0.9% solution of sodium chloride in wateris also referred to as “physiological saline”. It has the same osmoticpressure as blood serum and is therefore suitable as an isotonicsolution for intravenous injection or infusion. Any other osmoticallyeffective substances may also be used for isotonization as long as theyare physiologically safe and well tolerated, such as glucose, glucosesubstitutes (fructose, sorbitol, xylitol) or glycerol. In anotherpreferred embodiment, the pharmaceutical formulations may additionallycontain further plasma-adapted electrolytes. The preparation of suchisotonic formulations is known to the skilled person. An example of anisotonic solution with plasma-adapted electrolytes is the so-calledTyrode solution. It contains 0.8 g of NaCl, 0.02 g of KCl, 0.02 g ofCaCl₂, 0.01 g of MgCl₂, 0.005 g of NaH₂PO₄, 0.1 g of NaHCO₃ and 0.1 g ofglucose in 100 ml of distilled water. Another example is the so-calledRinger solution which contains 0.8% sodium chloride, 0.02% potassiumchloride, 0.02% calcium chloride and 0.1% sodium hydrogencarbonate. Ofcourse, the anions of the electrolytes may also be replaced bymetabolizable anions; thus, for example, the sodium hydrogencarbonate inthe Ringer solution may be replaced by 0.3 or 0.6% sodium lactate. Acorresponding electrolyte composition or solution is known to theskilled person as “Ringer lactate”. Further metabolizable anions whichmay be used alone or in combination are acetate (e.g., “Ringer acetate”)or malate.

In another embodiment of the invention, the pharmaceutical formulationsmay also be in the form of hypertonic solutions. Hypertonic solutionsare those having a higher osmotic pressure than that of the human blood.The application of hypertonic pharmaceutical formulations may beadvantageous in certain clinical pictures. The required high osmoticpressures of hypertonic solutions is adjusted by adding correspondingamounts of osmotically effective substances, e.g., by sodium chloride,which may be used in concentrations of up to 7.5% and more for thispurpose.

To avoid and reduce the risk of infections, the pharmaceuticalformulations according to the invention are preferably subjected tosterile filtration or heat sterilization. Particularly suitable for thesterile filtration of aqueous or colloidal aqueous pharmaceuticalformulations according to the invention are fine-pore filter cartridges,such as those provided by the company Sartorius under the trade nameSARTPORE. Such filter cartridges with a pore diameter of 0.2 μm aresuitable, for example. In addition, the pharmaceutical formulationsaccording to the invention may be subjected to heat sterilizationwithout the hydroxyethylstarches being adversely affected. Preferably,the heat sterilization is performed at a temperature above 100° C., morepreferably from 105 to 150° C., especially from 110 to 130° C., forexample, 121° C., for a period of up to 30 minutes, preferably up to 25minutes, especially from 18 to 22 minutes.

In a preferred embodiment, the pharmaceutical formulation is a volumereplacement. Volume replacements are used for replacing intravascularfluid in animal and human organisms. Volume replacements are used, inparticular, in the prophylaxis and therapy of hypovolemia. It is notcritical whether the hypovolemia results from the immediate loss ofblood or body fluids, such as in acute bleeding, traumas, surgery, burnsetc., or from disturbed distributions between macro- andmicrocirculation, such as in sepsis, or from vasodilation, such asduring the initiation of anesthesia. The volume replacements are furtherclassified into the so-called plasma replacements and the so-calledplasma expanders. For the plasma replacements, the intravascularlyapplied volume of the agent also corresponds to the volume supplied tothe vessels. In contrast, for the plasma expanders, the intravascularlyapplied liquid volume of the expander is lower than the volume actuallysupplied to the vessels. This phenomenon is based on the fact that theuse of plasma expanders disturbs the oncotic equilibrium between theintra- and extravascular spaces and additional liquid volume flows fromthe extravascular space into the vascular system to be treated.

Plasma expanders are distinguished from plasma replacements essentiallyby the fact that the concentration of the hydroxyethylstarches accordingto the invention contained therein is increased and/or the concentrationof the respective electrolytes causes an oncotic and/or osmoticimbalance.

The pharmaceutical formulation according to the invention may furthercontain a pharmaceutically active ingredient or combinations of activeingredients and thus serve as a medium for administering the activeingredients dissolved therein, especially by injection and infusion.

The present invention further relates to the use of a pharmaceuticalformulation according to the invention for the preparation of a volumereplacement or plasma replacement or plasma expander.

More preferably, the pharmaceutical formulations according to theinvention may be used as a volume replacement or plasma replacement orplasma expander. Preferably, the pharmaceutical formulations serve formaintaining normovolemia. The maintaining of normovolemia is ofparticular importance for hemodynamic stability, which has a criticalinfluence on the human or animal organism, for example, with respect tothe blood pressure, the diuresis rate or the heart rate. In order tocompensate a loss of intravascular liquid as quickly as possible andrestore normovolemia, the pharmaceutical formulations according to theinvention have proven particularly advantageous, because as compared tothe plasma replacements known in the prior art, especially low-molecularweight HES solutions, such as HES 130/0.4, they have an extended plasmahalf life, especially in the critical phase immediately after infusion.The pharmaceutical formulations according to the invention are alsoadvantageous, in particular, because it has surprisingly been found thatthe blood and/or plasma viscosity is not increased when the compositionsare used, in contrast to the statement made in U.S. Pat. No. 5,502,043for high-molecular weight HES, and because blood coagulation isinhibited less as compared to other high-molecular weight formulations.The fact that the plasma viscosity surprisingly is not increased alsoprovides for an improvement of microcirculation and for an improvednutritive oxygen supply to the vessels.

The invention further relates to the use of the pharmaceuticalformulation according to the invention for maintaining normovolemiaand/or for improving the macro- and microcirculation and/or forimproving the nutritive oxygen supply and/or for stabilizinghemodynamics and/or for improving the volume efficiency and/or forreducing the plasma viscosity and/or for increasing anemia toleranceand/or for hemodilution, especially for therapeutic hemodilution indisturbed blood supply and arterial, especially peripheral arterial,occlusive diseases.

The pharmaceutical formulations according to the invention or thehydroxyethylstarch according to the invention are preferably used forthe preparation of medicaments, especially medicaments for maintainingnormovolemia and/or for improving the macro- and microcirculation and/orfor improving the nutritive oxygen supply and/or for stabilizinghemodynamics and/or for improving the volume efficiency and/or forreducing the plasma viscosity and/or for increasing anemia toleranceand/or for hemodilution, especially for therapeutic hemodilution indisturbed blood supply and arterial, especially peripheral arterial,occlusive diseases.

In addition, the pharmaceutical formulations according to the inventionor the hydroxyethylstarches according to the invention areadvantageously employed in methods for treating the maintenance ofnormovolemia and/or for improving the macro- and microcirculation and/orfor improving the nutritive oxygen supply and/or for stabilizinghemodynamics and/or for improving the volume efficiency and/or forreducing the plasma viscosity and/or for increasing anemia toleranceand/or for hemodilution, especially for therapeutic hemodilution indisturbed blood supply and arterial, especially peripheral arterial,occlusive diseases.

The present invention further relates to a kit comprising separately:

(i) a hydroxyethylstarch according to the invention;

(ii) a sterile salt solution, preferably sodium chloride solution; andoptionally

(iii) a pharmaceutically active ingredient or a combination of activeingredients.

In a preferred embodiment, the kit according to the invention includesthe individual components (i), (ii) and optionally (iii) in separatedcompartments in a multi-compartment bag, wherein all components may beseparated, or certain components, such as (i) and (ii), may be containedtogether in one compartment.

The invention is further illustrated by the following Examples.

PREPARATION EXAMPLES FOR HES RAW MATERIALS Example 1 Preparation of HESRaw Materials with Identical MS and C₂/C₆ Ratios but Different MolecularWeights

The HES species described in the experimental part for the in vivostudies were prepared from one reaction charge by fractional hydrolysis.For this purpose, the following procedure was adopted. With vigorousstirring, 30 kg of thin boiling waxy maize starch was suspended in 52.2kg of wfi (water for injection) at room temperature. In order to hydratethe starch optimally, the suspension was subsequently gelatinated byheating it to at least 85° C. After repeatedly inertizing the suspensionby sparging with nitrogen for 10 min followed by evacuation, the starchwas activated by adding 5.1 kg of NaOH. Subsequently, 4.159 kg of cooledethylene oxide in liquid form was introduced into the reactor, and thetemperature was slowly increased to 40° C., and the reaction mixture wasleft at that temperature for 2 hours with constant stirring. Unreactedethylene oxide was removed from the reaction charge by repeatedlyinertizing as described above. Then, three HES preparations withidentical MS and C₂/C₆ ratios but different Mw were prepared from thisraw HES by stepwise acidic hydrolysis. For reducing the molecularweight, the solution was adjusted to pH 2.0 with 20% HCl, heated up to75±1° C. and left at that temperature until the average molecular weightMw of the HES colloid as determined by means of GPC-MALLS had decreasedto 865 kD. One third of the hydrolysis charge was removed from thereactor and immediately cooled down to a temperature of below 50° C.After the solution was decolorized by treatment with active charcoal,the solution was filtered by means of commercially available prefiltersand sterile filters and, after being diluted to 12% by ultrafiltration(UF), purified. Thus, polyethersulfone membranes of the companyMillipore with a cut-off of 10 kD were used. In the course of the UF,the Mw is slightly increased due to the partial elimination of thelow-molecular weight HES fraction. This increase depends on the startingMw of the colloid preparation but mainly on the declared cut-off of theUF membrane employed and the UF membrane lot employed. In order toachieve a desired target molecular weight after the UF, the Mw shiftduring the UF must be preliminarily established experimentally with theUF membrane lot employed. It is also to be noted that the hydrolysiscontinues from the time of sampling for determining the Mw during theacidic hydrolysis until the established Mw value has been obtained.Therefore, the decrease of the Mw is to be monitored systematicallythroughout the hydrolysis period, and the time at which the target Mwwill be reached is to be estimated by extrapolation of the Mw over time.Then, the hydrolysis is stopped at this extrapolated time. Thehydrolysis charge remaining after the first third has been removed wascontinued in the meantime until the average molecular weight Mw haddecreased to 460 kD. Subsequently, the second third was processed in thesame way as the first third. In parallel, the remaining third wasfurther hydrolyzed to a Mw of 95 kD and subjected to the same processingprocedure as the other two partial charges. From partial charge 1, HES900/0.42 (C₂/C₆ ratio=4.83) could be obtained, from partial charge 2,HES 500/0.42 (C₂/C₆ ratio=4.83) could be obtained, and from partialcharge 3, HES 130/0.42 (C₂/C₆ ratio=4.83) could be obtained.

After the ultrafiltration was completed, the colloid concentration wasadjusted to 6%, and the pH value to 5.5, the solution was isotonized byadding NaCl, filled in glass bottles at 500 ml each, and sterilized at121° C. for 20 minutes.

Example 2 Preparation of Further HES Raw Materials

In order to produce HES colloids with other molar substitutions andC₂/C₆ ratios, a number of further experiments were performed on the samescale, the amount of ethylene oxide being varied accordingly. Inaddition, the acidic hydrolysis was stopped when different Mw (targetMw) were reached. These experiments are summarized in the followingTable 1: TABLE 1 Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Reaction conditionsStarch (kg) 30.0 30.0 30.0 30.0 30.0 WFI (kg) 52.0 52.0 52.0 52.0 52.0NaOH 50% (kg) 9.5 4.7 4.7 9.5 4.7 Ethylene oxide (kg) 4.0 3.6 4.4 4.53.8 Target Mw (after acidic 1500 990 885 770 665 hydrolysis) (kDa) HEScharacteristics MS (mol/mol) 0.39 0.40 0.45 0.46 0.42 Mw after UF (kDa)1520 1050 915 795 710 C₂:C₆ ratio 2.3 6.0 5.9 2.2 6.1

Example 3 Influence of the Molar Ratio of NaOH to Starch during theHydroxyethylation on the C₂/C₆ Ratio

In order to demonstrate the controllability of the C₂/C₆ ratio by themolar ratio of NaOH to anhydroglucose units of the starch, 30 kg of thinboiling waxy maize starch was admixed with different amounts of NaOH andreacted with ethylene oxide at 40° C. In Table 2, the amounts ofreagents employed and the C₂/C₆ ratios as well as the MS of the HESproducts obtained by this reaction are listed. As can be seen, the C₂/C₆ratio decreases as the ratio of NaOH to starch increases. This is due tothe fact that the base-catalyzed hydroxyethylation of the starch at alow NaOH concentration is preferably effected at the hydroxyethyl groupsin 2 position of the anhydroglucose units, which are the most reactive.By higher NaOH concentrations, the C₆ hydroxy groups, which are per seless reactive, are also activated sufficiently to become efficientlyhydroxyethylated. TABLE 2 Control of the C₂/C₆ ratio duringhydroxyethylation NaOH Molar (50% by ratio of WFI Starch weight) NaOH toEthylene Trial [kg] [kg] [kg] starch oxide [kg] MS C₂/C₆ # 1 50 30 1.50.1 4.0 0.4 12 # 2 50 30 4.5 0.3 3.8 0.4 7 # 3 50 30 11.1 0.75 4.4 0.4 3

PREPARATIONS EXAMPLES OF HES FINISHED PRODUCTS

In the following Table 3, the formulations for the preparation ofvarious HES solutions are stated. The HES was employed as an HESconcentrate after ultrafiltration. The amount of HES concentraterequired for the preparation of a 6% or 10% HES solution was determinedby rule-of-three calculation. Another possibility is to use spray-driedHES, which does not pose any problem to the skilled person who has aspraying tower at his disposal. The HES employed had a molecular weightof 900 kD and an MS of 0.42.

In a 200 l reaction tank, the respectively required amount of HESconcentrate and the amounts of salts and NaOH solution as stated in theTable were weighed, and the salts were dissolved with stirring. Afteradjusting the pH of solutions 1, 4, 5, 7 and 8 to 5.5 and of solutions2, 3, 6 to 6.0, water for injection (WFI) was added in such an amountthat the theoretical Na concentrations according to the specificationwere reached.

As obvious to the skilled person, the formulations can be varied widelyby changing the proportions of the stated active ingredients orauxiliary agents and by omitting or adding further substances, and ifother HES species are used, corresponding solutions can be prepared withthose as well. TABLE 3 1 2 3 4 5 6 7 8 Preparation formulations HES[g/l] 60 60 60 60 100 100 100 100 NaCl [g/l] 9.00 6.369 17.37 100.0 9.006.369 30.00 60.00 KCl [g/l] 0 0.373 0.373 0 0 0.373 0 0 MgCl₂ [g/l] 00.203 0.203 0 0 0.203 0 0 CaCl₂ [g/l] 0 0.294 0.294 0 0 0.294 0 0 Naacetate 0 5.032 5.032 0 0 5.032 0 0 [g/l] Results of analyses of thesolutions HES [%] 6.0 6.1 6.0 5.9 10.0 10.1 9.9 10.0 Na [mM] 153.9 146.1334.3 1711 153.7 146.0 512.5 1026 K [mM] 0 5.1 5.0 0 0 5.0 0 0 Mg [mM] 01.0 0.9 0 0 1.0 0 0 Ca [mM] 0 2.1 2.0 0 0 2.0 0 0 Cl [mM] 154.8 120.2308.5 1712 154.8 120.1 513.7 1028 Acetate [mM] 0 37.0 37.0 0 0 37.0 0 0pH 5.5 6.0 5.9 5.3 5.4 6.1 5.4 5.5

MEASURING METHODS OF THE APPLICATION EXAMPLES

In the following, the measuring methods are described with which theblood and plasma samples were examined.

Native Blood Measurements:

Blood samples with added citrate were treated in the laboratory asfollows:

One sample was used immediately for the measurement of blood viscosity(Rheostress® 1, Thermo-Haake, Karlsruhe, Germany) with linearlyincreasing shear rates of 1 to 240 per second. The viscosity wasexamined at shear rates of 1 per second and 128 per second. Beforeanalysis on a Thromboelastograph® (TEG®, Haemoscope Corporation, Niles,Ill.), the blood samples were incubated in a water bath of 37° C. forone hour. The blood recalcification and the TEG® measurements wereperformed in accordance with the manufacturer's instructions. Thecoagulation index (CI), which summarizes several partial functions ofthromboelastography, was determined.

Plasma Measurements:

The blood samples were centrifuged at 4° C. and 3000 rpm (Rotana/RP,Hettich, Bäch, Switzerland) for 15 minutes. The plasma viscosity wasmeasured as described above for the blood determination.

The prothrombin time (PT) and the activated partial thromboplastin time(aPTT) were determined by means of an automated coagulation analyzer(BCS, Dade Behring, Marburg, Germany) using a PT reagent containing arecombinant tissue factor (Innovin®, Dade Behring) and an aPTT reagentcontaining ellagic acid (Actin FS®, Dade Behring). PT values wereconverted to INR values based on the ISI values supplied by themanufacturer. The functional activity of von Willebrand factor (vWF) wasdetermined by means of a commercial ristocetin cofactor assay (vWF RCA,Dade Behring) in an automated coagulation analyzer (BCS, Dade Behring).The vWF activity was established by the ability to agglutinate humanthrombocytes in the presence of ristocetin. Agglutination was determinedby means of turbidity measurements with a coagulation analyzer. AntigenvWF was detected by a commercial ELISA kit (Asserachrom vWF antigenic,Roche Diagnostics, Rotkreuz, Switzerland) in accordance with themanufacturer's instructions.

The HES concentration was quantified after extraction from blood plasmaand hydrolysis into glucose monomer units (H. Forster et al.,Infusionstherapie 1981; 2: 88-94). The plasma samples (1 ml) wereincubated at 100° C. for 60 minutes after being admixed with 0.5 ml ofKOH solutions 35% (w/w) (Fluka, Buchs, Switzerland). The HES wasprecipitated by adding 10 ml of ice-cold absolute ethanol (Fluka, Buchs,Switzerland) to the supernatant of the reaction mixture, followed byacid hydrolysis in 2 N HCl (Fluka, Buchs, Switzerland) for 60 minutes at100° C. The glucose determination was effected by using an enzyme testkit based on hexokinase/glucose 6-phosphatase (Boehringer Mannheim,Darmstadt, Germany).

The calculation of the pharmacokinetic parameters was effected with theassumption of a two-compartment model with a constant infusion rateusing the actual dosages and infusions periods (WinNonlin, Version 4.1,Pharsight Corp., Mountainview, Calif.).

Statistical Analysis:

The values were stated as mean value ± standard deviation. The two HESsolutions having a high molecular weight (500 and 900 kD) were comparedwith the low-molecular weight (130 kD) solution by means of the JMP 5.1statistics package (SAS Institute, Inc., Cary, N.C.). The interaction ofsolution and time effects was tested by means of two-sided ANOVAanalysis taking into account the Bonferroni correction. For thestatistical analysis of the pharmacokinetic parameters, Student'sunpaired t test was used. A p of <0.05 was considered statisticallysignificant.

APPLICATION EXAMPLES

For the in vivo experiments described below, hydroxyethylstarchesaccording to the invention having average molecular weights (Mw) of500,000 and 900,000 Daltons and identical molar substitutions (MS=0.42)and identical C₂/C₆ ratios (4.83) (in the following Application Examplesreferred to as HES 500/0.42 and HES 900/0.42, respectively) were used(see Preparation Example for HES raw materials). Bothhydroxyethylstarches (HES 900/0.42 and HES 500/0.42) were dissolved in0.9% saline at a concentration of 6% using 0.2 μm filter cartridges(Sartpore; Sartorius), subjected to sterile filtration, filled in glassbottles and heat-sterilized at 121° C. for 15 minutes. A low-molecularweight hydroxyethylstarch (Mw=130,000 Daltons) with identical MS andC₂/C₆ ratio (in the following Application Examples referred to as HES130/0.42), which was also in 6% concentration in 0.9% saline, served asa comparative solution. As described, it was obtained from the samereaction charge as the high-molecular weight starches according to theinvention, from which it was therefore distinguished only by themolecular weight.

Examination of Plasma Elimination and its Influence on Blood Coagulation

30 pigs were randomized in 3 groups of 10 animals each. One groupreceived an intravenous infusion of HES 900/0.42, another received aninfusion of HES 500/0.42, and the third received an infusion of HES130/0.42 for comparison. In all cases, the dose was 20 ml/kg body weightof the HES solutions, which were 6% each, and the infusion took 30minutes. For the infusion and the subsequent blood sampling, the animalswere anesthetized (halothane anesthesia) and subjected to controlledrespiration. Blood samplings were effected before the beginning ofinfusion, after 5, 20, 40, 60, 120 and 240 minutes as well as 24 hoursafter the end of the infusion. In the blood samples and plasma samplesobtained therefrom, the following parameters were determined: blood andplasma viscosities, HES concentration, prothrombin time, partialthromboplastin time, von Willebrand factor, factor VIII and ristocetincofactor as well as the usual thromboelastographic characteristics. Fromthe course of the HES concentrations from the end of the infusion until24 h thereafter, the area under the concentration vs. time curve (=AUC,area under the curve), the α and β half lives and the clearance werecalculated. The calculation of AUC was effected according to thelog-linear trapezoidal rule, and the calculation of the remainingpharmacokinetic parameters was based on a two compartment model. Thisyields 2 half lives, α and β, the α half life designating the transitionof the HES from the central compartment (corresponds essentially to theintravascular space) into the peripheral compartment, and the β halflife designating the back distribution in the reverse direction.

The course of the HES concentration and the pharmacokinetic parametersshowed a longer plasma residence time of the high-molecular weightvariants (HES 900/0.42 and HES 500/0.42) as compared to thelow-molecular weight HES (HES 130/0.42). Thus, the AUCs and α half liveswere significantly larger or longer, respectively, in the high-molecularweight variants as compared to the lower-molecular weight control;accordingly, the clearance of the high-molecular weight HES types wassignificantly lower than that of the lower-molecular weight HES.

However, surprisingly and completely unlike previously known types ofmedium- or high-molecular weight HES (HES 200/0.5; HES 200/0.6; HES450/0.7), there was no relevant differences in the plasma concentrationat the time “24 hours after infusion” between the high-molecular weightvariants according to the invention and the low-molecular weightcomparative solution (cf. FIG. 1). This means that the high-molecularweight hydroxyethylstarches according to the invention have asignificantly longer plasma residence time as compared to thelower-molecular weight comparative HES in the phase immediately afterthe infusion, which is critical to volume efficiency but that they haveno tendency to accumulation in the circulation, in contrast topreviously known high-molecular weight HES types. Instead, the HESvariants according to the invention, like the lower-molecularcomparative HES, had virtually completely disappeared from thecirculation 24 hours after the infusion. TABLE 4 Table 4: Area under theconcentration vs. time curve (AUC), clearance (CL), α and β half lives(t_(1/2α) and t_(1/2β)) after infusion of 20 ml/kg each of 6% HES130/0.42, 6% HES 500/0.42 and 6% HES 900/0.42 in pigs. Solution HES130/0.42 HES 500/0.42 HES 900/0.42 AUC (g · min/l) 1156 ± 223   1542 ±142** 1701 ± 321** CL (ml/min) 39.1 ± 7.9  30.1 ± 5.4* 26.0 ± 4.1**t_(1/2α) (min) 39.9 ± 10.7 53.8 ± 8.6* 57.1 ± 12.3* t_(1/2β) (min) 331.8± 100.0 380.6 ± 63.3  379.9 ± 75.8 The significance test was effected between HES 500 and HES 900 each incomparison with HES 130/0.42 by means of a Student's unpaired t test;*p < 0.01;**p < 0.001.

Table 4: Area under the concentration vs. time curve (AUC), clearance(CL), α and β half lives (t_(1/2α) and t_(1/2β)) after infusion of 20ml/kg each of 6% HES 130/0.42, 6% HES 500/0.42 and 6% HES 900/0.42 inpigs.

The significance test was effected between HES 500 and HES 900 each incomparison with HES 130/0.42 by means of a Student's unpaired t test; *:p<0.01; **: p<0.001.

The high-molecular weight HES species (HES 500/0.42 and HES 900/0.42)showed significantly larger areas under the concentration vs. time curve(AUC), corresponding to a longer residence time in the intravascularspace, significantly longer initial plasma half lives (t_(1/2α)) andsignificantly lower clearance rates as compared to lower-molecularweight ones (HES 130/0.42).

FIG. 1 shows the course of the concentration of low-molecular weight HES(HES 130/0.42) and high-molecular weight HES (HES 500/0.42 and ES900/0.42) in the plasma after infusion of 20 ml/kg of the respective HESsolution in pigs. Initially, HES 500/0.42 and HES 900/0.42 wereeliminated more slowly from the intravascular space as compared to HES130/0.42 (see also Table 4: pharmacokinetic parameters); however, in theend phase of elimination, i.e., 24 h after the end of the infusion,there was no longer a relevant difference between the plasmaconcentrations (the average concentrations were below 0.2 g/l, which iswithin the range of the determination limit).

Thus, it has been found that the hydroxyethylstarches according to theinvention on the one hand have a longer initial plasma half life ascompared to currently known lower-molecular weight reference solutions(HES 130/0.42) but on the other hand can be eliminated from thecirculation within 24 hours after infusion as readily as the latterlower-molecular weight comparative preparations, which are advantageousin this respect.

Also, the coagulation analyses performed (plasmatic coagulation tests,thromboelastography, determination of vWF concentrations) yieldedunexpected results, because the results were completely different fromthose which could be achieved previously with high-molecular weight HESpreparations. While already medium-molecular weight and, to a much morepronounced extent, high-molecular weight hydroxyethylstarches usuallyhave previously shown a far stronger impairment of blood coagulation interms of hypocoagulability as compared to lower-molecular weight HESsolutions (J. Treib et al., Intensive Care Med. (1999), pp. 258 to 268;R. G. Strauss et al., Transfusion (1988), pp. 257-260), no significantdifferences were found between the high-molecular weight HESpreparations according to the invention and the known lower-molecularweight comparative solution (cf. Table 5). TABLE 5 after infusion afterinfusion after infusion after infusion after infusion Parameter Solutionbefore infusion 5 min 1 hour 2 hours 4 hours 24 hours p value PT (s) HES130/0.42  8.57 ± 0.73  9.15 ± 0.66  9.00 ± 0.70  9.06 ± 0.67  8.77 ±0.42  9.30 ± 0.64 — HES 500/0.42  9.32 ± 1.90  9.08 ± 0.49  8.97 ± 0.56 8.80 ± 0.33  8.57 ± 0.37  9.38 ± 0.58 0.533 HES 900/0.42  8.59 ± 1.06 8.66 ± 0.51  8.56 ± 0.56  8.40 ± 0.27  8.21 ± 0.27  9.42 ± 0.51 0.068aPTT HES 130/0.42 12.19 ± 1.83 14.19 ± 4.21 13.68 ± 3.57 13.59 ± 1.5713.11 ± 1.10 14.58 ± 3.01 — (s) HES 500/0.42 11.97 ± 1.00 13.37 ± 1.1212.65 ± 0.73 13.04 ± 1.31 13.17 ± 1.31 13.72 ± 1.40 0.893 HES 900/0.4211.94 ± 1.31 12.59 ± 1.16 13.10 ± 1.62 12.87 ± 1.06 12.78 ± 2.02 13.37 ±1.50 0.706 CI HES 130/0.42  4.53 ± 1.33  3.30 ± 0.91  4.18 ± 0.91  4.81± 0.63  4.48 ± 0.65  5.22 ± 0.35 — HES 500/0.42  5.04 ± 0.81  2.88 ±1.28  4.47 ± 0.77  4.71 ± 0.65  4.18 ± 0.98  5.14 ± 0.79 0.303 HES900/0.42  5.35 ± 0.79  3.28 ± 1.18  4.47 ± 0.93  4.93 ± 0.82  4.68 ±1.03  5.53 ± 0.55 0.468 vWF HES 130/0.42 33.41 ± 10.18 30.09 ± 7.6832.20 ± 8.63 33.59 ± 10.94 40.91 ± 10.48 28.80 ± 6.84 — funct. HES500/0.42 32.11 ± 4.85 25.38 ± 3.84 27.65 ± 4.32 30.80 ± 4.67 37.94 ±4.65 23.04 ± 4.19 0.853 (%) HES 900/0.42 30.48 ± 3.95 24.78 ± 3.35 27.14± 4.14 31.41 ± 4.83 37.87 ± 8.06 24.84 ± 6.74 0.768 vWF HES 130/0.4246.10 ± 8.45 35.80 ± 4.83 39.40 ± 8.42 40.10 ± 9.21 44.30 ± 6.80 38.22 ±5.54 — antig. HES 500/0.42 42.80 ± 4.13 37.90 ± 7.37 35.50 ± 4.22 37.60± 7.41 39.80 ± 10.65 38.00 ± 8.15 0.499 (%) HES 900/0.42 48.10 ± 19.5633.30 ± 5.48 35.70 ± 5.89 37.80 ± 4.26 42.00 ± 4.85 41.67 ± 6.08 0.473

Table 5 shows the time course of the plasmatic coagulation parametersprothrombin time (PT), activated partial thromboplastin time (aPTT),functional activity of von Willebrand factor (vWF functional) andantigenic concentration of von Willebrand factor (vWF antigenic) and thetime course of the coagulation index (CI) of thromboelastography afterinfusion of 20 ml/kg each of 6% HES 130/0.42, 6% HES 500/0.42 and 6% HES900/0.42, respectively, in pigs. The test for statistic significance(interaction of solution and time effects) was performed between HES500/0.42 and HES 900/0.42, respectively as compared to HES 130/0.42, bymeans of two-sided ANOVA. Despite higher molecular weights, higherconcentrations and a longer residence time in plasma (cf. FIG. 1 andTable 4), the high-molecular weight HES species according to theinvention (HES 500/0.42 and HES 900/0.42) did not influence bloodcoagulation to any higher extent as low-molecular weight HES (HES130/0.42).

In other words, despite a longer plasma residence time achieved byincreasing the molecular weight, the hydroxyethylstarches according tothe invention did not show the drawbacks of known high-molecular weightsolutions, such as the impairment of blood coagulation conferred bythem.

In addition, it was surprisingly found in the animal experimentalstudies that the high-molecular weight hydroxyethylstarches according tothe invention did not increase the blood and plasma viscosity ascompared with lower-molecular weight HES, in contrast to knownhigh-molecular weight hydroxyethylstarches. At low shear forces, a lowerviscosity was even found among the hydroxyethylstarches according to theinvention than among the lower-molecular weight HES (cf. Table 6: plasmaviscosity). TABLE 6 Parameter Solution before infusion after infusion 5min after infusion 20 min after infusion 40 min Plasma visc. HES130/0.42 0.016383 ± 0.008241 0.012721 ± 0.005245 0.012620 ± 0.0042800.012875 ± 0.003349 γ = 1/s HES 500/0.42 0.012313 ± 0.005196 0.012582 ±0.001873 0.013591 ± 0.001912 0.012936 ± 0.002555 (mPas) HES 900/0.420.016869 ± 0.007240 0.012375 ± 0.002487 0.015985 ± 0.005659 0.015968 ±0.006499 Plasma visc. HES 130/0.42 0.001208 ± 0.000097 0.001327 ±0.000644 0.001138 ± 0.000089 0.001167 ± 0.000105 γ = 128/s HES 500/0.420.001246 ± 0.000212 0.001110 ± 0.000045 0.001125 ± 0.000058 0.001106 ±0.000037 (mPas) HES 900/0.42 0.001271 ± 0.000078 0.001141 ± 0.0000640.001174 ± 0.000065 0.001166 ± 0.000075 Parameter Solution afterinfusion 1 hour after infusion 2 hours after infusion 4 hours afterinfusion 24 hours p value Plasma visc. HES 130/0.42 0.014928 ± 0.0039440.018318 ± 0.006364 0.015097 ± 0.007475 0.024577 ± 0.008847 — γ = 1/sHES 500/0.42 0.013070 ± 0.002860 0.011384 ± 0.005949 0.013736 ± 0.0039740.015434 ± 0.004320 0.005 (mPas) HES 900/0.42 0.013678 ± 0.0022520.014217 ± 0.003234 0.013557 ± 0.003713 0.015556 ± 0.004308 0.003 Plasmavisc. HES 130/0.42 0.001142 ± 0.000084 0.001272 ± 0.000217 0.001232 ±0.000136 0.001229 ± 0.000102 — γ = 128/s HES 500/0.42 0.001112 ±0.000048 0.001206 ± 0.000114 0.001183 ± 0.000112 0.001257 ± 0.0000680.386 (mPas) HES 900/0.42 0.001146 ± 0.000055 0.001304 ± 0.0002240.001228 ± 0.000096 0.001247 ± 0.000104 0.444

Table 6 shows the time course of plasma viscosity at low and high shearforces (γ=1/s and γ=128/s after infusion of 20 ml/kg each of 6% HES130/0.42, 6% HES 500/0.42 and 6% HES 900/0.42, respectively, in pigs.The test for statistic significance (interaction of solution and timeeffects) was performed between HES 500/0.42 and HES 900/0.42,respectively as compared to HES 130/0.42, by means of two-sided ANOVAand did not show any differences between the high-molecular weight HESspecies (HES 900 and HES 500) and the lower-molecular weight HES (HES130). At lower shear forces, the interaction between the solution andtime effects in the plasma viscosity proved to be lower among thehigh-molecular weight HES species (HES 500/0.42 and HES 900/0.42) ascompared to the lower-molecular weight HES (HES 130/0.42). However, thiswas due to the time rather than the solution effect. At high shearforces, there was no difference; in particular, the plasma viscosity wasnot higher among the high-molecular weight HES species (HES 900/0.42 andHES 500/0.42) than among lower-molecular weight HES (HES 130/0.42).

Thus, the plasma viscosity does not increase among the HES solutionsaccording to the invention. The fact that no increase of plasmaviscosity was observed is surprising because hydroxyethylstarch with acomparable molecular weight of 500,000 as disclosed in U.S. Pat. No.5,502,043 (Comparative Example 3) exhibited an increase of plasmaviscosity. If the viscosity is not increased, this leads to undisturbedcapillary perfusion (microcirculation) and an improved nutritive oxygensupply to the tissues.

In addition to the above described in vivo studies, in vitro studieswere also performed in which the influence of the C₂/C₆ ratio on bloodcoagulation was especially tested, i.e., also by means ofthromboelastography. For this purpose, three high-molecular weight HESsolutions (Mw: 800 kD) with a low MS (0.4) and a low (3:1), medium (7:1)or high (12:1) C₂/C₆ ratio were prepared and examined as follows. From30 male and female surgical patients (exclusion criteria: knowncoagulation disorders, treatment with blood coagulation inhibitors,ingestion of acetylsalicylic acid or other non-steroidalanti-inflammatory medicaments within 5 days before the surgery), a bloodsample was taken during the initiation of anesthesia. In every bloodsample, the coagulation as measured by means of thromboelastography,i.e., in undiluted blood and after in vitro hemodilution (20%, 40% and60%) with each of three HES solutions (HES 800/0.4/3:1; HES 800/0.4/7:1and HES 800/0.4/12:1). As with the in vivo studies, the parameterdetermined was the coagulation index (CI), which summarizes theindividual partial functions of thromboelastography. The mean values(±SD) of the CI values found are represented in the following Table 7,i.e., as deviation from the CI in the respective undiluted blood sample.TABLE 7 HES HES HES 800/0.4/3:1 800/0.4/7:1 800/0.4/12:1 20%hemodilution −2.32 −2.86 −3.35 (1.20) (1.22) (1.07) 40% hemodilution−5.91 −6.17 −6.50 (1.68) (2.03) (1.69) 60% hemodilution −9.71 −10.37−10.55 (2.70) (3.05) (2.60)

In all hemodilution stages, the CI relative to the CI of the nativeblood was decreased the less, i.e., the blood coagulation influenced theless, the lower the C₂/C₆ ratio of the HES used for hemodilution was.The solution effect between the hemodilution series was significantlydifferent (p<0.05; ANOVA). The results show that a decrease of the C₂/C₆ratio of hydroxyethylstarches is favorable for their influence on bloodcoagulation, namely in such terms that blood coagulation is inhibitedless at a lower C₂/C₆ ratio as compared to a high one. This is importantbecause HES solutions are employed, inter alia, as plasma replacementsafter traumatically or surgically caused bleedings, and in thissituation, they must not add to the blood loss by inhibiting bloodcoagulation. The results of the above described study further show thatthe C₂/C₆ ratio of HES solutions has an intrinsic effect on bloodcoagulation which is independent of other molecular parameters of HESand their behavior in the circulation. This has not been known to date.

1. A hydroxyethylstarch having an average molecular weight, Mw, ofgreater than or equal to 500,000, characterized by having a molarsubstitution MS of from 0.25 to 0.5 and a C₂/C₆ ratio of from 2 to below8. 2-23. (canceled)
 24. The hydroxyethylstarch according to claim 1,wherein the molar substitution MS is from 0.35 to 0.5, preferably from0.39 to smaller than or equal to 0.45, especially from greater than 0.4to 0.44.
 25. The hydroxyethylstarch according to claim 1, wherein theaverage molecular weight is from above 600,000 to 1,500,000, preferablyfrom 620,000 to 1,200,000, more preferably from 700,000 to 1,000,000.26. The hydroxyethylstarch according to claim 1, wherein the said C₂/C₆ratio is from 2 to 7, preferably from 2.5 to smaller than or equal to 7,more preferably from 2.5 to 6, even more preferably from 4 to
 6. 27. Thehydroxyethylstarch according to claim 1, wherein the hydroxyethylstarchis obtainable from a waxy maize starch.
 28. The hydroxyethylstarchaccording to claim 24, wherein the average molecular weight is fromabove 600,000 to 1,500,000, preferably from 620,000 to 1,200,000, morepreferably from 700,000 to 1,000,000.
 29. The hydroxyethylstarchaccording to claim 24, wherein the C₂/C₆ ratio is from 2 to 7,preferably from 2.5 to smaller than or equal to 7, more preferably from2.5 to 6, even more preferably from 4 to
 6. 30. The hydroxyethylstarchaccording to claim 25, wherein the C₂/C₆ ratio is from 2 to 7,preferably from 2.5 to smaller than or equal to 7, more preferably from2.5 to 6, even more preferably from 4 to
 6. 31. The hydroxyethylstarchaccording to claim 24, wherein the hydroxyethylstarch isobtainable froma waxy maize starch.
 32. The hydroxyethylstarch according to claim 25,wherein the hydroxyethylstarch is obtainable from a waxy maize starch.33. The hydroxyethylstarch according to claim 26, wherein thehydroxyethylstarch is obtainable from a waxy maize starch.
 34. Apharmaceutical formulation comprising a hydroxyethylstarch comprising anaverage molecular weight, Mw, of greater than or equal to 500,000, amolar substitution MS of from 0.25 to 0.5 and a C₂/C₆ ratio of from 2 tobelow
 8. 35. The pharmaceutical formulation according to claim 34,wherein the pharmaceutical formulation isin the form of at least one ofan aqueous solution and a colloidal aqueous solution.
 36. Thepharmaceutical formulation according to claim 34, wherein thehydroxyethylstarch in the formulation is in a concentration of up to20%, preferably from 0.5 to 15%, more preferably from 2 to 12%.
 37. Thepharmaceutical formulation according to claim 34, wherein thepharmaceutical formulation further comprises sodium chloride, preferablyin a concentration of 0.9%.
 38. The pharmaceutical formulation accordingto claim 34, wherein the pharmaceutical formulation further comprisesplasma-adapted electrolytes.
 39. The pharmaceutical formulationaccording to claim 34, wherein the pharmaceutical formulation is in theform of at least one of a buffered solution and a solution withmetabolizable anions.
 40. The pharmaceutical formulation according toclaim 34, wherein the pharmaceutical formulation is in the form of ahypertonic solution.
 41. The pharmaceutical formulation according toclaim 34, wherein the hydroxyethylstarch is at least one of sterilefiltered and heat sterilized.
 42. The pharmaceutical formulationaccording to claim 34, characterized by being a volume replacement. 43.The pharmaceutical formulation according to claim 34, further comprisingat least one pharmaceutically active ingredient.
 44. The pharmaceuticalformulation according to claim 35, wherein the hydroxyethylstarch is ina concentration of up to 20%, preferably from 0.5 to 15%, morepreferably from 2 to 12%.
 45. The pharmaceutical formulation accordingto claim 35, further comprising sodium chloride.
 46. The pharmaceuticalformulation according to claim 35, further comprising plasma-adaptedelectrolytes.
 47. The pharmaceutical formulation according to claim 35,wherein the pharmaceutical formulation is in the form of at least one ofa buffered solution and a solution with metabolizable anions.
 48. Thepharmaceutical formulation according to claim 35, wherein thepharmaceutical solution is in the form of a hypertonic solution.
 49. Thepharmaceutical formulation according to claim 35, wherein thehydroxyethylstarch is at least one of sterile filtered and heatsterilized.
 50. The pharmaceutical formulation according to claim 35,characterized by being a volume replacement.
 51. The pharmaceuticalformulation according to claim 35, further comprising at least onepharmaceutically active ingredient.
 52. A method of preparing a plasmareplacement or plasma expander, said method comprising the step ofpreparing a pharmaceutical formulation comprising a hydroxyethylstarchcomprising an average molecular weight, Mw, of greater than or equal to500,000, characterized by having a molar substitution MS of from 0.25 to0.5 and a C₂/C₆ ratio of from 2 to below
 8. 53. The method of claim 52,wherein the pharmaceutical formulation is in the form of at least one ofan aqueous solution and a colloidal aqueous solution.
 54. The method ofclaim 52, wherein the hydroxyethylstarch is in a concentration of up to20%, preferably from 0.5 to 15%, more preferably from 2 to 12%.
 55. Themethod of claim 52, further comprising sodium chloride, preferably in aconcentration of 0.9% in the pharmaceutical formulation.
 56. The methodof claim 52, further comprising plasma-adapted electrolytes.
 57. Themethod of claim 52, wherein the pharmaceutical formulation is in theform of at least one of a buffered solution and a solution withmetabolizable anions.
 58. The method of claim 52, wherein thepharmaceutical formulation is in a form of a hypertonic solution. 59.The method of claim 52, wherein the hydroxyethylstarch is at least oneof sterile filtered and heat sterilized.
 60. The method of claim 52,wherein the pharmaceutical formulation is used as a volume replacement.61. The method of claim 52, wherein further comprising at least onepharmaceutically active ingredient.
 62. A process for preparing ahydroxyethylstarch comprising the steps: (i) reacting water-suspendedstarch with ethylene oxide; and (ii) partially hydrolyzing a starchderivative with acid until a desired range of average molecular weightof the hydroxyethylstarch is reached; and wherein the hydroxyethylstarchcomprises an average molecular weight, Mw, of greater than or equal to500,000, characterized by having a molar substitution MS of from 0.25 to0.5 and a C₂/C₆ ratio of from 2 to below
 8. 63. The process according toclaim 62, wherein an alkalizing agent is added to said water-suspendedstarch.
 64. The process according to claim 62, wherein an alkalizingagent is added to said suspended starch in such an amount that a molarratio of alkalizing agent to starch is larger than 0.2, preferably from0.25 to 1, especially from 0.3 to 0.8.
 65. The process according toclaim 62, further comprising the steps of sterilization.
 66. The processaccording to claim 62, wherein the suspended starch is corn starch. 67.The process according to claim 62, wherein the acid is hydrochloricacid.
 68. The process according to claim 63, wherein the alkalizingagent is NaOH.
 69. The process according to claim 65, further comprisingthe step of ultrafiltration.
 70. Use of a pharmaceutical formulation forat least one of maintaining normovolemia, improving macro- andmicrocirculation, improving nutritive oxygen supply, stabilizinghemodynamics, improving volume efficiency, reducing plasma viscosity,increasing anemia tolerance, and for hemodilution, wherein thepharmaceutical formulation contains a hydroxyethylstarch having anaverage molecular weight, Mw, of greater than or equal to 500,000,characterized by having a molar substitution MS of from 0.25 to 0.5 anda C₂/C₆ ratio of from 2 to below 8, and introducing the pharmaceuticalformulation in a treatment process.
 71. The use of a pharmaceuticalformulation according to claim 70, wherein the hemodilution involvestherapeutic hemodilution in disturbed blood supply and arterialdiseases.
 72. The use of a pharmaceutical formulation according to claim71, wherein the arterial diseases involves peripheral arterial occlusivediseases.
 73. The use of a pharmaceutical formulation according to claim70, wherein the pharmaceutical formulation is in the form of at leastone of an aqueous solution and a colloidal aqueous solution.
 74. The useof a pharmaceutical formulation according to claim 70, wherein thehydroxyethylstarch is in a concentration of up to 20%, preferably from0.5 to 15%, more preferably from 2 to 12%.
 75. The use of apharmaceutical formulation according to claim 70, wherein thepharmaceutical formulation further contains a sodium chloride,preferably in a concentration of 0.9%.
 76. The use of a pharmaceuticalformulation according to claim 70, wherein the pharmaceuticalformulation further includes plasma-adapted electrolytes.
 77. The use ofa pharmaceutical formulation according to claim 70, wherein thepharmaceutical formulation is in the form of at least one of a bufferedsolution and a solution with metabolizable anions.
 78. The use of apharmaceutical formulation according to claim 70, wherein thepharmaceutical formulation is in the form of a hypertonic solution. 79.A kit comprising separately: (i) a hydroxyethylstarch; and (ii) asterile salt solution wherein the hydroxyethylstarch comprises anaverage molecular weight, Mw, of greater than or equal to 500,000,characterized by having a molar substitution MS of from 0.25 to 0.5 anda C₂/C₆ ratio of from 2 to below
 8. 80. The kit of claim 79, wherein thesterile salt solution is sodium chloride solution.
 81. The kit of claim79, further comprising at least one pharmaceutically active ingredient.82. The kit of claim 79, wherein the molar substitution MS is from 0.35to 0.5, preferably from 0.39 to smaller than or equal to 0.45,especially from greater than 0.4 to 0.44.
 83. The kit of claim 79,wherein the average molecular weight is from above 600,000 to 1,500,000,preferably from 620,000 to 1,200,000, more preferably from 700,000 to1,000,000.
 84. The kit of claim 79, wherein the C₂/C₆ ratio is from 2 to7, preferably from 2.5 to smaller than or equal to 7, more preferablyfrom 2.5 to 6, even more preferably from 4 to
 6. 85. The kit of claim79, wherein the average molecular weight is from above 600,000 to1,500,000, preferably from 620,000 to 1,200,000, more preferably from700,000 to 1,000,000.
 86. The kit of claim 79, wherein thehydroxyethylstarch and the sterile salt solution are in separatedcompartments in a multi-compartment bag.
 87. The kit of claim 81,wherein the hydroxyethylstarch, the sterile salt solution, and the atleast one pharmaceutically active ingredient are in separatedcompartments in a multi-compartment bag.